Microparticulate packings

Fig. 3.1b lists some of the properties of two commercially available microparticulate packings. The first is a 5 /im unmodified silica, the second a 3 fim silica, chemically modified with octadecylsilane (ODS, C-18). 

You twill find as you get further involved with hplc that there is a bewildering Zariety of microparticulate packings available in the trade literature. The textbook by Hamilton and Sewell (second Edition, Chapter 4) has an extensive list, as do most other textbooks. To keep things in perspective, remember that almost all the work in... 

Currently, almost all the available SEC columns are packed with the high efficiency, microparticulate packings (<10u). Recent state-of-the-art developments on column packings have been described by Majors (10). A listing of such type of packing materials is shown in Table 1. 

One can see three routes to an increase in the value of resolution First, one can increase the plate number N which results in an increase in resolution proportional to the square root of N The use of microparticulate packings was a major breakthrough in this respect If we assume, however, that the chromatographer already has at his disposal a high efficiency column, then he must go to great... 

Fresh solutions of the standards are prepared in the solvent used as chromatographic eluent. Calibration solutions should be as dilute as possible, in order to avoid any concentration dependence of sample retention volumes. The concentration effect causes an increase of retention volumes with increased sample concentration. As a rule of thumb, when high efficiency microparticulate packings are used, the concentration of narrow standards should be < 0.025% (w/v) for MW over 10 , < 0.05% for MW between 10 and 2 x 10 , and < 0.1% for MW down to lOl With a lower MW and in the oligomer range, the sample concentration can be higher than the previously suggested values. 

Figure 21.14. Comparison of columns used in liquid chromatography. A Classical open column chromatography with large porous particle packings B HFLC with pellicular packings C HPLC with microparticulate packings.

Short (3-10 cm) HPLC columns can also be used for rapid sample concentration. Microbore reversed-phase columns have been used for preconcentration of proteins prior to microsequence analysis, after collection and dilution of milliliter fractions from conventional HPLC columns [36]. Gradient elution at low flow rates (0.1-0.2 mL/min for 2-mm-ID or 0.02-0.04 mL/min for 1-mm-ID columns) permits recovery of proteins in volumes as small as 25 pL, with concentration factors up to 80-foId. The high capacities of porous microparticulate packings enable 50-100 pg to be loaded onto microbore columns as long as the sample is introduced in a weak solvent. 

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